We have continued to characterize factors involved in regulating melanocyte function in the skin. The specific aims of this project include: (1) Identify and characterize factors that regulate melanocyte function;(2) Characterize trafficking mechanisms of melanosomal proteins and their disruption in pigmentary diseases;(3) Elucidate downstream effects of MC1R signaling by its agonists and antagonists;and (4) Characterize the regulation of melanocyte function by UV radiation. Regulation at the Subcellular Level - We have shown that TYR, TRP1 (now TYRP1) and TRP2 (now DCT) have distinct catalytic functions and interact within a complex in melanosomes. TYR has the critical catalytic function in melanogenesis, i.e. the hydroxylation of tyrosine to DOPA, but TYRP1 and DCT have important post-TYR functions that modify the type of melanins synthesized. Those post-TYR reactions not only optimize the physical characteristics of melanins produced and maximize their photoprotective benefits, but they also minimize the inherent cytotoxicity of melanogenic precursors and their by-products. Mutations in the genes encoding TYRP1 or DCT often result in premature melanocyte death, presumably from cytotoxic intermediates. We have characterized the sorting signals, processing and trafficking pathways of all 3 TRPs as well as the structural proteins involved in melanosome maturation. We have shown that Pmel17/gp100 (and its chaperone MART1) plays an important role in the maturation of vesicular Stage I melanosomes to the fibrillar Stage II form. That transition is essential to the subsequent targeting and delivery of melanogenic enzymes to melanosomes and is required for melanin synthesis and deposition to occur. We have shown that Pmel17 undergoes an extremely complex series of glycosylations and can traffic via a number of distinct sorting pathways to reach melanosomes (and other subcellular compartments). Our studies have shown that the Pro-Ser-Thr rich repeat domain of Pmel17 (called RPT) is essential for the formation of fibrils;although deletion of the RPT domain does not affect Pmel17 trafficking, fibril formation is disrupted. Mutations in Pmel17 can be quite toxic and in mice the gene is called silver because follicular melanocytes die and the hair becomes prematurely gray. Regulation at the Cellular Level - Factors such as UV, DKK1, MSH and ASP, regulate the quantity and quality of melanins produced in the skin by melanocytes. We have detailed molecular changes in gene expression, and mechanisms involved in those changes, which occur in response to various physiological stimuli. The G-protein coupled melanocortin receptor 1 (MC1R) is regulated by its agonist MSH and antagonist ASP. Those interactions regulate skin and hair color, as well as responses to the environment. Our research on MC1R has involved characterizing: (1) MC1R genotype and melanocyte phenotype, (2) mechanisms involved with MC1R activation or inhibition by MSH or ASP, (3) downstream events that regulate MC1R function in determining melanocyte differentiation, and (4) the involvement of MC1R function in responses of melanocytes to other physiological stimuli, such as UV. The switch to produce yellow/red pheomelanin or black/brown eumelanin is under the control of the MC1R and its ligands. We have also elucidated the involvement of dynein and spectrin with the molecular motors required to move melanosomes from the perinuclear area of melanocytes to the peripheral dendrites, which is critical for their eventual transfer to keratinocytes. Our recent microarray studies have characterized the changes in gene expression patterns elicited by ASP or MSH in a time-dependent manner, which has revealed the wide range of genes affected by MC1R function, including those involved in development, migration and other non-pigmentary functions. Regulation at the Tissue Level - We have also characterized the influence of dermal fibroblasts on skin pigmentation. Melanocyte density and pigmentation in palmoplantar human skin (i.e. skin on the palms and soles) is 5X less than that found elsewhere on the body (i.e. non-palmoplantar skin). We have shown that fibroblasts derived from palmoplantar skin significantly suppress the growth and pigmentation of melanocytes compared with non-palmoplantar fibroblasts, and that this is due to their secretion of DKK1, an inhibitor of canonical Wnt signaling. We demonstrated that DKK1 dramatically inhibits melanocyte function, probably through beta-catenin-mediated regulation of MITF which in turn modulates the growth and differentiation of melanocytes. These results explain why skin on the palms and soles is hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in palmoplantar areas during embryogenesis. Treatment of keratinocytes with DKK1 increases their proliferation and decreases their melanin uptake, whereas treatment of melanocytes with DKK1 decreases their growth and production of melanin pigment. In sum, our studies have elucidated why human skin is thicker and paler on the palms and soles than on the trunk through topographical/site-specific differences in DKK1 secretion by dermal fibroblasts which affects the overlying epidermis. We have also recently identified neuregulin-1 (NRG1), another factor secreted by fibroblasts in the dermis, that plays a key role in regulating the constitutive pigmentation of skin phenotypes ranging from very light in color to very dark. Given the importance of the melanin content in the skin to photoprotection against UV damage and subsequent photocarcinogenesis, further studies are underway to determine whether the NRG1 ligand and/or its receptor on melanocytes can be targeted to modulate skin color.